The integration of high-throughput single-cell profiling technologies with RNA velocity analysis has enabled the reconstruction of dynamic cellular differentiation trajectories at unprecedented resolution. Despite these advances, current visualization techniques for RNA velocity are predominantly confined to 2-dimensional representations, typically employing arrows or streamlines. While effective for depicting simple cellular trajectories, these approaches are insufficient for capturing the complex topologies of multipartite cellular transitions. This limitation highlights the need for advanced 3-dimensional visualization tools that can more accurately convey the structure and dynamics of velocity-inferred transitions in single-cell data. Here, we present Cell Journey, an interactive visualization platform specifically developed for 3-dimensional analysis and representation of RNA velocity trajectories derived from single-cell datasets. The platform features an intuitive graphical interface supporting both unimodal and multimodal data, accommodates multiple input formats, and provides extensive customization capabilities for trajectory visualization. Cell Journey computes RNA velocity vector fields on a user-defined 3-dimensional grid and constructs velocity trajectories using either Euler integration or the fourth-order Runge-Kutta method. The platform enables dynamic exploration of cellular dynamics through interactive visual elements, including streamlines, streamlets, cones, and volumetric plots. Furthermore, it allows users to investigate changes in feature activity along selected paths, facilitating deeper insights into cellular state transitions within complex multimodal single-cell datasets.

The simultaneous profiling of diverse molecular modalities offers unprecedented insight into complex biological processes, yet it poses significant computational challenges. Here, we introduce mTopic, a generalizable topic modeling framework for analysis of unlimited types of modalities across both single-cell and spatial contexts. mTopic enables identification of coherent multimodal molecular programs at enhanced resolution and supports investigation of cross-modality associations. ### Competing Interest Statement P.R. and M.T. are named inventors on European Patent application EP25224154.2 relating to the work of this manuscript. Foundation for Polish Science, FENG.02.01- IP.05-T005/23 National Science Center, 2022/45/N/NZ2/02311, 2022/46/E/NZ2/0037

Aims/Purpose: Excessive levels of the intracellular second messengers Ca2+ and cAMP have been linked with photoreceptor cell death during retinal degeneration (RD). We investigated if a combination (TMB) of common clinical drugs; tamsulosin and metoprolol (alpha‐ and beta‐adrenergic antagonists, Gq‐ and Gs‐coupled, respectively), and bromocriptine (a D2‐like dopamine‐receptor agonist, Gi‐coupled), that inhibit intracellular Ca2+ and cAMP signaling could be repurposed to mitigate RD.Methods: Drug efficacy was tested in four distinct RD models: rd10, P23H, and Rpe65−/− mice; and PDE6A−/− dogs. The duration of the drug trials ranged from 1‐wk to 7‐months. Drug serum levels were measured by liquid chromatography‐mass spectrometry (LC‐MS). We used primarily photopic and scotopic electroretinography (ERG) and optical coherence tomography (OCT) to assess drug efficacy. Molecular biology methods such as immunohistochemistry, immunoblotting, and RNA‐sequencing (bulk and single cell) were used to document therapeutic mechanisms, as well as to confirm therapeutic effects.Results: Dietary TMB improved cone function and slowed cone degeneration in P23H mice. In rd10 mice, both rod and cone function were significantly improved by TMB; and cone degeneration was significantly slowed. In dark‐reared rd10 mice, the drug efficacy was associated with decreased lipid peroxidation preceding the onset of cone degeneration. Dietary TMB improved retinal function and optomotor responses in Rpe65−/− mice but did not halt rod or cone degeneration. Seven‐month‐long subcutaneous sustained infusion of TMB into PDE6A−/− dogs led to higher cone counts at the end of the trial. TMB mitigated forskolin‐induced cAMP activity in an ex vivo retina preparation.Conclusions: Our results suggest that simultaneous inhibition of Gs‐ and Gq‐coupled receptors and activation of Gi‐coupled receptors by a combination of existing drugs is a viable therapeutic strategy for RD.

The neural crest (NC) is a transient embryonic structure composed of highly migratory multipotent stem cells that generate diverse cell types and orchestrate early neurovascular patterning. It has long been assumed that NC cells are exhausted after development once their progeny fully differentiate. However, through NC lineage tracing, single-cell and spatial transcriptomics, interactome modeling, and in vivo imaging, we identified a population of NC-derived multipotent (not lineage-restricted) cells persisting within the adult mouse leptomeninges. Following ischemic stroke, loss- and gain-of-function analyses revealed that these cells are reactivated and recruited toward injured vascular endothelium via SDF1α-CXCR4 signaling; undergo stromal cell transition within the perivascular niche, regulated by β-catenin and STAT3 pathways; and restore vascular integrity through pleiotrophin-mediated signaling. These findings suggest that the adult leptomeninges harbor a vestigial reservoir of NC-derived multipotent cells—once central to embryogenesis and vasculogenesis—that can be re-invoked to promote neurovascular repair after cerebral injury.

The recent expansion of single-cell technologies has enabled simultaneous genome-wide measurements of multiple modalities in the same single cell. The potential to jointly profile such modalities as gene expression, chromatin accessibility, protein epitopes, or multiple histone modifications at single-cell resolution represents a compelling opportunity to study developmental processes at multiple layers of gene regulation. Here, we present Ocelli, a lightweight Python package implemented in Ray for scalable visualization and analysis of developmental multimodal single-cell data. The core functionality of Ocelli focuses on diffusion-based modeling of biological processes involving cell state transitions. Ocelli addresses common tasks in single-cell data analysis, such as visualization of cells on a low-dimensional embedding that preserves the continuity of the developmental progression of cells, identification of rare and transient cell states, integration with trajectory inference algorithms, and imputation of undetected feature counts. Extensive benchmarking shows that Ocelli outperforms existing methods regarding computational time and quality of the reconstructed low-dimensional representation of developmental data.

PURPOSE. Homeostatic plasticity is crucial for maintaining stable neural activity by adjusting strength and intrinsic properties of synapses. This mechanism is vital for normal nervous system function and plays a role in various neurological conditions, including retinal degenerations. Sensitive night vision has been shown in P23H/Gnat2-/- retinitis pigmentosa (RP) mice, which lack cone phototransduction and rely solely on rods, even after losing more than half of their rod photoreceptors. While homeostatic plasticity has been proposed as a potential explanation, the underlying molecular mechanisms remain unclear. The aim of this study was to investigate the molecular basis of this phenomenon. METHODS. Single cell RNA-sequencing (scRNA-seq) and bulk retina proteomics were used to investigate the transcriptomic and proteomic changes of the degenerating retinas in 1-month-old P23H/Gnat2-/- RP and Gnat2-/-control mice. Immunohistochemistry was used to analyze the expression of synaptic SNARE complex and vesicle proteins, SNAP25 and SYT1, in the outer plexiform layer, the site of rod axon terminals. RESULTS. This study shows a significant upregulation of genes encoding synaptic SNARE complex and vesicle proteins (Snap25, Stxbp1, and Syt1) in P23H mouse rods. Bulk retina proteomics analysis shows trends toward upregulation of the corresponding proteins as well as upregulation of many matrix-associated and trans-synaptic-complex proteins. Immunohistochemistry shows persistent SYT1 and SNAP25 expression in the outer plexiform layer of P23H/Gnat2-/- mice despite significant rod death. CONCLUSIONS. Rod degeneration induces molecular changes in the P23H/Gnat2-/- mouse rods that suggest synaptic plasticity and strengthening of rod-rod bipolar cell synaptic transmission in early RP.

Aims/Purpose: Photoreceptor death triggers retinal rewiring and remodeling that are generally thought to be detrimental for vision. Some recent research results challenge this dogma. It was recently shown that in early‐stage retinitis pigmentosa (RP), the retina compensates for the reduced rod population activity by upscaling synaptic transmission between the remaining rods and rod bipolar cells. The purpose of the current project is to study the molecular mechanism behind this phenomenon.Methods: To model the primary pathology, P23H mouse model of autosomal dominant RP was used. Notably, wild‐type mice were not used as controls, instead, cone‐transducin (GNAT2) knockout mice that lack cone photoreceptor functionality were used. To produce “rod‐function‐only” RP mice, P23H and Gnat2‐/‐ mice were cross‐bred. Single cell RNA‐sequencing (scRNA‐seq; Gnat2‐/‐, n = 4; P23H/Gnat2‐/‐, n = 4) and retinal global proteomics (Gnat2‐/‐, n = 5; P23H/Gnat2‐/‐, n = 10) analyses were performed at P30. Statistical significance was set at q < 0.05. Additional protein expression analyses for synaptic proteins were performed using immunohistochemistry and immunoblotting.Results: ScRNA‐seq analysis revealed upregulation of several major ribbon synapse genes exclusively in P23H/Gnat2‐/‐ mouse rods. These included genes that are crucial in presynaptic structure and neurotransmitter release, such as the Ca2+ sensor Syt1, snare protein complex component Snap25, ribbon protein Ctbp2, and the synaptic vesicle protein Sv2b. In addition, upregulation of the glutamate transporter Slc1a2 was found. Based on proteomics data, STX1B and SYT7 were overexpressed in whole P23H/Gnat2‐/‐ mouse retinas as compared to controls.Conclusions: Early‐stage RP in mice associates with increased expression of several rod ribbon synapse components that play a role in glutamate release. These results imply that regulation of presynaptic glutamate release may play a role in improving retinal synaptic transmission during RP.

AbstractInherited retinopathies are devastating diseases that in most cases lack treatment options. Disease-modifying therapies that mitigate pathophysiology regardless of the underlying genetic lesion are desirable due to the diversity of mutations found in such diseases. We tested a systems pharmacology-based strategy that suppresses intracellular cAMP and Ca2+ activity via G protein-coupled receptor (GPCR) modulation using tamsulosin, metoprolol, and bromocriptine coadministration. The treatment improves cone photoreceptor function and slows degeneration in Pde6βrd10 and RhoP23H/WT retinitis pigmentosa mice. Cone degeneration is modestly mitigated after a 7-month-long drug infusion in PDE6A-/- dogs. The treatment also improves rod pathway function in an Rpe65-/- mouse model of Leber congenital amaurosis but does not protect from cone degeneration. RNA-sequencing analyses indicate improved metabolic function in drug-treated Rpe65-/- and rd10 mice. Our data show that catecholaminergic GPCR drug combinations that modify second messenger levels via multiple receptor actions provide a potential disease-modifying therapy against retinal degeneration.

MicroRNAs (miRs) are short, evolutionarily conserved non-coding RNAs that canonically downregulate expression of target genes. The miR family composed of miR-204 and miR-211 is among the most highly expressed in the retinal pigment epithelium (RPE) in both mouse and human, and also retains high sequence identity. To assess the role of this miR family in the developed mouse eye, we generated two floxed conditional knockout mouse lines crossed to the RPE65-ERT2-Cre driver mouse line to perform an RPE-specific conditional knockout of this miR family in adult mice. After Cre-mediated deletion, we observed retinal structural changes by optical coherence tomography; dysfunction and loss of photoreceptors by retinal imaging; and retinal inflammation marked by subretinal infiltration of immune cells by imaging and immunostaining. Single-cell RNA sequencing of diseased RPE and retinas showed potential miR-regulated target genes, as well as changes in non-coding RNAs in the RPE, rod photoreceptors, and Müller glia. This work thus highlights the role of miR-204 and miR-211 in maintaining RPE function and how the loss of miRs in the RPE exerts effects on the neural retina, leading to inflammation and retinal degeneration.

Mutations in the adiponectin receptor 1 gene (AdipoR1) lead to retinitis pigmentosa and are associated with age-related macular degeneration (AMD). This study explores the effects of AdipoR1 gene deficiency in mice, revealing a striking decline in ω3 polyunsaturated fatty acids (PUFA), an increase in ω6 FAs, and elevated ceramides in the retina. The AdipoR1 deficiency impairs peroxisome proliferator-activated receptor α (PPARα) signaling, which is crucial for FA metabolism, particularly affecting proteins associated with FA transport and oxidation in the retina and retinal pigmented epithelium (RPE). Our lipidomic and proteomic analyses indicate changes that could affect membrane composition and viscosity through altered ω3 PUFA transport and synthesis, suggesting a potential influence of AdipoR1 on these properties. Furthermore, we noted a reduction in the Bardet-Biedl syndrome (BBS) proteins, which are crucial for forming and maintaining photoreceptor outer segments that are PUFA-enriched ciliary structures. Diminution in BBS-proteins content combined with our electron microscopic observations raises the possibility that AdipoR1 deficiency might impair ciliary function. Treatment with inhibitors of ceramide synthesis led to substantial elevation of ω3 LC-PUFAs, alleviating photoreceptor degeneration and improving retinal function. These results serve as the proof of concept for a ceramide-targeted strategy to treat retinopathies linked to PUFA deficiency, including AMD.

Droplet microfluidic methods have massively increased the throughput of single-cell sequencing campaigns. The benefit of scale-up is, however, accompanied by increased background noise when processing challenging samples and the overall RNA capture efficiency is lower. These drawbacks stem from the lack of strategies to enrich for high-quality material or specific cell types at the moment of cell encapsulation and the absence of implementable multi-step enzymatic processes that increase capture. Here we alleviate both bottlenecks using fluorescence-activated droplet sorting to enrich for droplets that contain single viable cells, intact nuclei, fixed cells or target cell types and use reagent addition to droplets by picoinjection to perform multi-step lysis and reverse transcription. Our methodology increases gene detection rates fivefold, while reducing background noise by up to half. We harness these properties to deliver a high-quality molecular atlas of mouse brain development, despite starting with highly damaged input material, and provide an atlas of nascent RNA transcription during mouse organogenesis. Our method is broadly applicable to other droplet-based workflows to deliver sensitive and accurate single-cell profiling at a reduced cost.

Chronic, progressive retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa, arise from genetic and environmental perturbations of cellular and tissue homeostasis. These disruptions accumulate with repeated exposures to stress over time, leading to progressive visual impairment and, in many cases, legal blindness. Despite decades of research, therapeutic options for the millions of patients suffering from these disorders remain severely limited, especially for treating earlier stages of pathogenesis when the opportunity to preserve the retinal structure and visual function is greatest. To address this urgent, unmet medical need, we employed a systems pharmacology platform for therapeutic development. Through integrative single-cell transcriptomics, proteomics, and phosphoproteomics, we identified universal molecular mechanisms across distinct models of age-related and inherited retinal degenerations, characterized by impaired physiological resilience to stress. Here, we report that selective, targeted pharmacological inhibition of cyclic nucleotide phosphodiesterases (PDEs), which serve as critical regulatory nodes that modulate intracellular second messenger signaling pathways, stabilized the transcriptome, proteome, and phosphoproteome through downstream activation of protective mechanisms coupled with synergistic inhibition of degenerative processes. This therapeutic intervention enhanced resilience to acute and chronic forms of stress in the degenerating retina, thus preserving tissue structure and function across various models of age-related and inherited retinal disease. Taken together, these findings exemplify a systems pharmacology approach to drug discovery and development, revealing a new class of therapeutics with potential clinical utility in the treatment or prevention of the most common causes of blindness.

Mutations in many visual cycle enzymes in photoreceptors and retinal pigment epithelium (RPE) cells can lead to the chronic accumulation of toxic retinoid byproducts, which poison photoreceptors and the underlying RPE if left unchecked. Without a functional ATP-binding cassette, sub-family A, member 4 (ABCA4), there is an elevation of all-trans-retinal and prolonged buildup of all-trans-retinal adducts, resulting in a retinal degenerative disease known as Stargardt-1 disease. Even in this monogenic disorder, there is significant heterogeneity in the time to onset of symptoms among patients. Using a combination of molecular techniques, we studied Abca4 knockout (simulating human noncoding disease variants) and Abca4 knock-in mice (simulating human misfolded, catalytically inactive protein variants), which serve as models for Stargardt-1 disease. We compared the two strains to ascertain whether they exhibit differential responses to agents that affect cytokine signaling and/or ceramide metabolism, as alterations in either of these pathways can exacerbate retinal degenerative phenotypes. We found different degrees of responsiveness to maraviroc, a known immunomodulatory CCR5 antagonist, and to the ceramide-lowering agent AdipoRon, an agonist of the ADIPOR1 and ADIPOR2 receptors. The two strains also display different degrees of transcriptional deviation from matched WT controls. Our phenotypic comparison of the two distinct Abca4 mutant-mouse models sheds light on potential therapeutic avenues previously unexplored in the treatment of Stargardt disease and provides a surrogate assay for assessing the effectiveness for genome editing.

Dendritic cells (DCs) sense environmental cues and adopt either an immune-stimulatory or regulatory phenotype, thereby fine-tuning immune responses. Identifying endogenous regulators that determine DC function can thus inform the development of therapeutic strategies for modulating the immune response in different disease contexts. Tim-3 plays an important role in regulating immune responses by inhibiting the activation status and the T cell priming ability of DC in the setting of cancer. Bat3 is an adaptor protein that binds to the tail of Tim-3; therefore, we studied its role in regulating the functional status of DCs. In murine models of autoimmunity (experimental autoimmune encephalomyelitis) and cancer (MC38-OVA-implanted tumor), lack of Bat3 expression in DCs alters the T cell compartment-it decreases TH1, TH17 and cytotoxic effector cells, increases regulatory T cells, and exhausted CD8+ tumor-infiltrating lymphocytes, resulting in the attenuation of autoimmunity and acceleration of tumor growth. We found that Bat3 expression levels were differentially regulated by activating versus inhibitory stimuli in DCs, indicating a role for Bat3 in the functional calibration of DC phenotypes. Mechanistically, loss of Bat3 in DCs led to hyperactive unfolded protein response and redirected acetyl-coenzyme A to increase cell intrinsic steroidogenesis. The enhanced steroidogenesis in Bat3-deficient DC suppressed T cell response in a paracrine manner. Our findings identified Bat3 as an endogenous regulator of DC function, which has implications for DC-based immunotherapies.

The neural crest (NC) is a transient structure in vertebrate embryogenesis comprising highly migratory multipotent stem cells that give rise to a diverse array of cell types in organs throughout the body, including initiating neurovascular patterning. It is assumed that neural crest stem cells (NCSCs) disappear after development. Unexpectedly, using single-nucleus RNA-sequencing, we discovered residual quiescent NCSCs in the adult mouse meninges which are activated by injury and contribute to the brain’s homeostatic response. RNA velocity, pathway, and transcription factor analyses in a murine stroke model (combined with in vivo imaging) show that these adult NCSCs migrate towards the perivascular spaces of the infarct and undergo a perivascular stromal cell transition that is regulated by Ptp1b, Ghr, and Stat3. Loss- and gain-of-function experiments show that these “vestigial” NCSCs are required for restoring vascular endothelial barrier function via β-catenin and Stat3 signaling. These findings suggest that, in the adult, an unexpected reservoir of cells -- once pivotal to embryogenesis and vascular morphogenesis -- are re-invoked for neurovascular repair. ### Competing Interest Statement The authors have declared no competing interest.